Comparative Analysis of Sandeep Kumar, Antimicrobial Activity of … · 2015-06-19 · Climatic Zones of India Sandeep Kumar, Manila Yadav, Amita Yadav and Jaya Parkash Yadav Department

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ARCHIVES OF CLINICAL MICROBIOLOGYISSN 1989-8436

2015Vol. 6 No. 2:1

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Comparative Analysis of Antimicrobial Activity of

Methanolic Extracts of Aloe Vera and Quantification of Aloe- Emodin Collected From Different

Climatic Zones of India

Sandeep Kumar, Manila Yadav, Amita Yadav and Jaya Parkash Yadav

DepartmentofGenetics,MaharshiDayanandUniversity,Rohtak-124001,Haryana,India

Correspondencia: Jaya Parkash Yadav

DepartmentofGenetics,MaharshiDayanandUniversity,Rohtak-124001,Haryana,India

[email protected]

Ph: +919416474640

AbstractBackground: Aloe vera isaplantofLiliaceae familyhasbeentraditionallyusedforavarietyofmedicinalpurposes.Ithasmultipleapplicationssuchasemollient,purgative, antibacterial, antioxidant, antifungal, antiseptic and in cosmeticsindustries. The aim of present study was standardization of HPLC protocol forquantificationofaloe-emodinandstudyofantimicrobialactivityofmethanolicextracts of Aloe vera collected from different climatic regions of India againstpathogenicreferenceATCCstrains.

Methods and findings: Antibacterial and antifungal activity was checked byagarwelldiffusionmethod.HighPerformanceLiquidChromatographywasusedfor quantification of aloe- emodin from collected samples by comparing withstandard aloe-emodinmarker compound. The different accession ofAloe vera methanolicextractsrevealedsignificantantimicrobialactivityagainstthetestedstrains. Extracts were more active against gram negative bacteria than grampositiveones.Amountsofmarkercompoundaloe-emodinconcentrationvariedalotindifferentaccessions.Climaticconditionsshowednotableeffectonquantityofmarkercompound.

Conclusion: The present study has showed the role of climatic conditions andgeographical locations on the variation in amounts of aloe- emodin present indifferent crude extracts of Aloe vera due to presence of which it has showedstrongantimicrobialactivity.

Keywords: Aloe vera;Anthraquinone;Aloe-emodin;Antimicrobialactivity;MIC;Phytochemicals

Introduction Aloe vera is a shrubby, perennial succulent plant of Liliaceaefamily having turgid pea-green leaves joined at the stem in arosettepattern.Aloe vera plantischaracterizedbystemlesslarge,thick,fleshy leaveshavingasharpapexandaspinymargin[1].Aloeverahasbeentraditionallyusedforavarietyofmedicinalpurposes.ItisaninseparablepartofindigenousmedicinesystemofIndia.Ithasgainedhighimportanceforitsdiversetherapeuticproperties.Aloeveracontainsover75nutrientsand200active

compounds;includingvitamins,enzymes,minerals,sugars,lignin,anthraquinones, saponins, salicylic acidandaminoacidswhichareresponsiblefortheirmedicinalproperties[2]. Itssecondarymetabolites have multiple applications such as emollient,purgative,antibacterial,antioxidant,antifungal,antisepticandincosmeticsindustries[3-6].Aloeveragelhasbeenusedsinceearlytimesforthetopicaltreatmentofvariousskinconditionssuchascuts,burnsandeczema[7].Aloe verahasbeenshowntohaveantiinflammatoryactivity[8,9],immunostimulatoryactivity[10]and cell growth stimulatory activity [11]. Furthermore, activity

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2015ARCHIVES OF CLINICAL MICROBIOLOGYISSN 1989-8436 Vol. 6 No. 2:1

againstavarietyofinfectiousagentshasbeenattributedtoAloe vera;forinstanceantiviral[12]andantifungal[13].

Aloe veracontainsfreeanthraquinonesandtheirderivativeslikeBarbaloin, Aloe-emodin-9-anthrone, lsobarbaloin, Anthrone-C-glycosidesand chromones. In largeamounts these compoundsexert a powerful purgative effect while in smaller quantity;theyappeartoaidabsorptionfromtheguttherefore itactsaspotent antimicrobial agents [14]. Specific plant compoundssuch as anthraquinones [15,16] anddihydroxy- anthraquinones[17],aswellassaponins[18]havebeenproposedtohavedirectantimicrobialactivity.

Aloe- emodin (1,8-dihydroxy-3-(hydroxymethyl)anthracene-9,10-dione)derivedfromleavesofAloe veraisanantimicrobialcompound[19,20].Itisalsoknowntohaveanticancerousactivityinneuroectodermaltumors[21],lungsquamouscellcarcinoma[22], hepatoma cells [23], in a glia cell line [24] and a humangliomacell line [25,26].Present study focusesonantimicrobialactivity of methanolic extracts of Aloe vera from 12 differentaccessionscovering6agro-climaticregionsofIndia.Italsodealswith standardization of quantities of antimicrobial compoundaloe-emodinfromdifferentaccession.

Experimental SectionCollection of plant Samples were collected from 12 sites covering 6 agro-climaticzonesofIndia.Eachsitehad2sub-sites.Sampleswere collected in the months of Jan-Feb 2013. HealthyleavesofAloe vera werecollectedfromindividualplantsateachlocation.DifferentcollectionsiteshavebeendepictedinFigure 1.Geographical locationsandaveragerainfallofthesesiteshavebeengiveninTable 1.Theplantmaterialwas identified and authenticated by comparing theherbariumspecimenavailableinDepartmentofGenetics,M. D. University, Rohtak (India). Herbarium specimennumberisMDU-6803.Tissueswereplacedinsterileplasticbagsandbroughtinanicebox,sealedproperly.Allsampleswerebroughttothelaboratoryandprocessedfurther.

Preparation of Crude Plant MaterialsThecollectedleaveswerechoppedintosmallpiecesandshadedried at room temperature. 100 gm dried leaves pieces weregrindedandsoakedin60%ethanolsolutionfor24hrs.Sulphuricacidandchloroformwereaddedintotheextractiveandrefluxedtoremovethechloroformextractive[27].Theextractwaspreparedby cold percolation method. Stock solution was prepared bydissolvinginmethanolwiththehelpofrotaryshaker.Areddishbrowncolloidcontainingaloeextractivewasobtained,whichisfilteredbyusingWhatmanfilterpaper.Aftertheevaporationofthemethanol,ayellowish-browncolloidwasobtainedascrudeextract.

Antimicrobial EvaluationReference Strains: Pathogenic reference ATCC (AmericanTypeCultureCollection)strainswereusedforcheckingactivity.

9bacterialand2fungalstrainswereusedforthestudy.StrainswereprocuredfromPostGraduateInstituteofMedicalSciences,Rohtak(Haryana).India.Outof9bacterialstrains,7weregramnegativeviz. Shigella flexneriATCC12022, Proteus mirabilisATCC43071,Salmonella typhiATCC13311,Serratia marcescensATCC27137,Klebsiella pneumoniaATCC700603,Escherichia coliATCC25922,Pseudomonas aeruginosa ATCC27853and2weregrampositiveviz.Enterococcus faecalisATCC29212andStaphylococus aureusATCC259323.FungalstrainswereCandida albicans ATCC3018andAspergillus nigerATCC282).

In-vitro Antibacterial and Antifungal assayAntibacterial and antifungal activity was checked by agar welldiffusionmethod.100mgofthemethanolicextractwasdissolvedin1mlofDMSOandusedastestsample.Freshovernightgrownculture was used for inoculums preparation. Inoculums werepreparedinpeptonewaterandincubatedfor2hours.Turbiditywasadjustedequivalent to0.5McFarlandunits (approximately108 CFU/ml). Inoculums were spread over fresh nutrient agarplateswithasterilespreader.4wellsof6mmdiameterwerecutwiththehelpofasterilecutter.Controldiscwasplacedincentre.10,20,30and40µlofthispreparedtestsamplewasaddedtoallbacterialstrainsusedandDMSOwasusedasnegativecontrol.10µgstreptomycinand10µgketoconazolediscswereusedaspositive control respectively for antibacterial and antifungalactivity.BothwerepurchasedfromHimedialaboratoriesPvt.Ltd(India).Plateswereincubatedfor24hoursat37°C.ClearzoneofinhibitionaroundeachwellwasmeasuredwithhelpofstandardrulerHiAntibioticZoneScaleTM-CsuppliedbyHiMediaLaboratoriesPvt.Ltd.India.Eachexperimentwasdoneintriplicates.

Minimum Inhibitory Concentration [MIC]Minimum inhibitory concentration (MIC) is the lowestconcentration of an antimicrobial that will inhibit the visiblegrowthofamicroorganismafterovernightincubation.TheMIC

DifferentcollectionsitesofAloe verafromdifferentplacesofIndia

Figure 1

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values of extracts were determined based on a micro brothdilutionmethodin96multi-wellmicrotiterplatesdevelopedbySarkaretal.[28]withslightmodifications.50μlofnutrientbrothand50μlofnormalsalinewereaddedtoeachwellofplate.10μlofresazurinindicatorsolution(preparedbydissolvinga270mgtabletin40mlofsteriledistilledwater)wasaddedineachwell.Avolumeof100μloftestmaterials(stockconcentration10mg/mlofextracts)wasadded intothefirst rowof theplate.Serialdilutionswereperformedsuchthateachwellhadatotalof100μlofthetestmaterialinseriallydescendingconcentrations.Finally10 μl of bacterial suspension concentration of 5 x 106 CFU/mlwasaddedtoeachwell.Platehadacolumnwithstreptomycinaspositivecontrol.Theplateswerepreparedintriplicateandplacedin an incubator at 37°C for 18 to 24hours. Any colour changefrompurple to pink indicates growthofmicrobes. The highestdilutionatwhichnocolorchangeoccurredwastakenastheMICvalueofextractandwasexpressedinmg/ml.

HPLC Analysis Chemicals usedAloe-emodin (used for the control of retention times), HPLCgrade methanol, acetonitrile and phosphoric acid werepurchasedfromSigma-Aldrich(USA).ARgradeethanol,sulfuricacidandchloroformwereusedfortheextractpreparation.Ultrapurewater(18.2MΩ·cm−1)wasobtainedbymeansofaMilliQapparatus byMillipore, USA. Stock solutions of the analyte (1mg/mL)werepreparedbydissolvingpuresubstanceinmethanolandwerestableforatleasttwomonthswhenstoredat−20°C(asassessedbyHPLCassays).Standardsolutionswereprepareddailyby diluting stock solutions with themobile phase and directlyinjectedintotheHPLCsystem.

InstrumentationA High Performance Liquid Chromatography equipped withgradient elution capability, Ultraviolet spectrophotometer (UV)and photodiode array (PDA) as detector and an auto sampler(Agilent Technologies system, model 1260 Infinity, USA). AstainlesssteelC18column,lengthof250mm,internaldiameter4.6mmand5μmparticlesize(250mm×4.6mm5μm)columnwas used. The column temperature maintained at ambientcondition.

HPLC Analysis ConditionThemobilephaseconsistedofacetonitrile(A)and0.1%aqueousphosphoricacid(B)withagradientelutionof24%Aat0-12min,24-50%A at 12-22min, 50-24%A at 22-40min and 24%A at40-50 min. The flow rate was 1.0 ml/min and the separationwasmonitoredbyabsorbanceat254nm.Theinjectionvolumewas 20 μl. The identification of peakswas done by comparingHPLCchromatogramsofindividualswiththepeakofpurchasedstandardaloe-emodin.

ResultsMethanolic leaf extracts of Aloe vera were prepared. Yield ofextractshavebeenshowninTable 2.MaximumyieldwasobtainedfromPunjab(4.3gm)andHaryana(4.2gm)accessions.Minimum

yieldwasfromKerala(3.2gm).Allextractsshowedtheactivityagainst all reference bacterial and fungal strains. Antimicrobialactivitieswererangedfrom11.50mmto20.50mmasmeasuredbydiameterofzoneofinhibition.ResultofzoneofinhibitionofallaccessionsandcontrolagainstteststrainshavebeengiveninTable 3.AntibacterialactivityforsomeoftheA.veraaccessionshavebeengiveninFigure 2.

MaximumantibacterialactivitywasnotedagainstE.coliwithazoneofinhibitionof20.50mmandminimumactivitywasagainstP.aeruginosawith11.83mmzoneofinhibition(Table 3,Figure 2).ActivityagainstShigellaflexneriKlebsiellapneumoniaeandE.coliwasquietgoodascomparedtocontrol.ExtractsweremoreactiveagainstfungusC.albicansthanA.niger.Punjab,HaryanaandWestBengalaccessionsshowedgoodactivityagainstmostofthetestedstrains.SignificantactivitywasreportedagainstK.pneumoniawithamaximum16mmzoneofinhibitionbyWestBengalaccession.Controlshowedonly12mmzoneofinhibitionagainst this bacteria. Minimum activity was shown against P.

S.No. Accessions names

Place of collection Latitude Longitude

Average rainfall (mm)

1 Jammu Jammu 34°44'N 78°54'E 1,011

2 HimachalPradesh Palampur 32°29'N 78°10'E 1,251

3 Punjab Sangrur 30°40'N 75°50'E 6494 Haryana Rohtak 30°30'N 76°60'E 6175 Rajasthan Jaisalmair 27°00'N 74°00'E 209.56 Gujarat Gandhinagar 23°.00'N 72°.00'E 1,107

7 UttarPradesh Pratapgarh 27°40'N 80°00'E 904

8 MadhyaPradesh Bhopal 23°30'N 80°00'E 1,146

9 WestBengal Kolkata 23°00'N 87°00'E 1,582

10 AndhraPradesh Hyderabad 16°00'N 80°00'E 812.5

11 Goa Kochi 15°00'N 74°25'E 3,055

12 Kerala Vasco 10°00'N 74°06'E 3,005

Table 1 Geographical locationsandaverage rainfall ofplant collectionsites

Agro-climatic zone Name of State Yield (g)Highland

Jammu 4HimachalPradesh(HP) 3.8

Semi-arid

Haryana 4.2Punjab 4.3

Arid

Rajasthan 3.6Gujarat 3.5

HumidSubtropical

UttarPradesh(UP) 4MadhyaPradesh(MP) 3.7

Tropicalwet&dry

WestBengal(WB) 4.1AndhraPradesh(AP) 3.9

Tropicalwet

Goa 3.4Kerala 3.2

Table 2 YieldofAloe veraextractscollectedfromdifferentAgro-climaticzones



Accessions CA AN SF EF SA PM ST SM KP EC PAJammu 20.00±10 17.83±0.7 14.66±0.5 14.00±0.5 16.83±0.7 14.66±0.5 16.00±0.5 14.16±0.7 13.50±0.5 15.16±0.7 13.00±1.0HP 18.16±07 18.0±0.5 15.83±0.7 14.66±0.7 16.33±0.5 14.83±0.7 15.00±0.5 12.50±0.5 14.00±0.5 16.00±1.0 12.50±0.5Punjab 17.00±05 16.16±0.7 16.00±1.0 14.33±0.5 20.00±0.5 13.33±0.5 18.50±0.5 13.00±0.5 12.16±0.2 19.00±0.5 13.83±0.7Haryana 19.00±0.5 15.66±0.5 16.16±0.7 13.00±0.5 19.00±1.0 16.50±0.5 19.00±0.5 14.16±0.2 13.00±0.5 20.50±1.0 14.50±0.5Rajasthan 19.83±0.7 17.33±0.5 18.00±1.0 16.00±1.0 17.33±0.5 15.5±0.5 17.16±0.2 13.83±0.7 16.16±0.2 18.50±0.5 14.66±0.5Gujarat 17.16±0.7 16.50±0.5 14.33±0.5 14.00±1.0 17.00±1.0 13.00±0.5 18.5±0.5 13.50±0.5 13.33±0.7 19.83±0.7 13.66±0.5UP 19.50±0.5 15.00±1.0 14.66±0.5 13.5±0.5 20.16±0.2 14.33±0.5 17.33±0.5 14.33±0.5 13.50±0.5 18.33±0.5 14.00±1.0MP 18.00±1.0 11.5±0.5 16.00±0.5 14.0±0.5 18.5±0.5 14.33±0.5 15.83±0.7 12.50±0.5 14.50±0.5 19.00±1.0 12.50±0.5WB 17.66±0.5 16.0±1.0 16.00±1.0 12.66±0.5 19.16±0.7 13.00±0.5 18.16±0.2 13.33±0.7 16.00±1.0 20.50±0.5 13.50±1.0AP 19.00±1.0 17.50±0.5 16.83±0.7 13.66±0.5 20.83±0.7 12.83±0.7 17.33±0.7 14.00±0.5 14.83±0.7 19.5±1.0 14.00±1.0Goa 18.66±0.5 16.66±0.5 16.00±0.5 12.00±1.0 20.33±0.5 13.00±0.5 17.00±0.5 13.50±0.5 14.33±0.7 19.00±1.0 11.83±0.7Kerala 17.83±0.7 15.00±0.5 16.16±0.7 13.83±0.7 20.00±1.0 14.83±0.7 18.50±0.5 14.50±0.5 14.00±1.0 19.00±0.5 13.00±1.0Control 18.0±1.0 23.0±0.5 14.0±1.0 23.0±0.5 26.0±1.0 23.5±0.5 24.0±0.5 25.5±0.5 12.0±1.0 13.0±1.0 18.0±1.0Fungalstrains=CA-Candida albicans andAN-Aspergillus nigerBacterialstrains=EC-Escherichia coli,PM-Proteus mirabilis,KP- Klebsiella pneumoniae,PA-Pseudomonas aeruginosa,SF-Shigella flexneri,SM-Serratia marcescens,ST-Salmonella typhi,EF-Enterococcus faecalisandSA- Staphylococus aureusControlforfungalstrains=Ketocanozole(10µg)Controlforbacterialstrains=Streptomycin(10µg)

Table 3 AntimicrobialactivityofdifferentaccessionsofAloe veraCrudeextractsmeasuredbyzoneofinhibition(mm±SD)

Figure 2 PlateshowingzoneofinhibitionshownbymethanoliccrudeextractsofAloe vera;(a)RajasthanaccessionagainstEnterococcus faecalis;(b)HaryanaaccessionagainstEscherichia coli;(c)U.PaccessionagainstPseudomonas aeruginosa;(d)A.P.accessionagainstStaphylococcus aureus;(e)W.B.accessionagainstKlebsiella pneumonia;(f)PunjabaccessionSalmonella typhi;(g)RajasthanaccessionagainstProteus mirabilis;(h)JammuAccessionagainstEscherichia coli

aeruginosa, S.marcescens, S. typhi, P.mirabilis and E. faecalisas compared with control. Jammu and HP accessions showedgoodantifungalactivity.Extractsweremoreactiveagainstgramnegative bacteria than gram positive ones.MIC values rangedfrom1.2mg/mlto5mg/ml.MinimumMICvalueof1.2mg/mlwas found against E. coli revealed by most of the accessions.Maximum MIC was against Proteus mirabilis, PseudomonasaeruginosaandfungusA.nigershownbymostoftheaccessions.MICvaluesofalltheaccessionshavebeenshowninTable 4.

TheHPLCanalysisrevealedthattherewasmarkabledifferencein the quantity of aloe- emodin from different accessions thatwere collected throughout India on thebasis of their differentclimatic zones. The results obtained indicated the presence ofanthraquinone derivative aloe- emodin in each accessionwiththesignificantdifferencesintheirquantities.Importantaspectslike Sample name, Climatic zone, Retention time (RT), Areaof peak and Yield percentages of compound (aloe- emodin) ofHPLCanalysisaregivenintheTable 5.HPLCchromatogramsofstandardalongwith3accessionsvizGujarat,UttarPradeshandHaryanahavebeenshowninFigures 3a-3d.

RT’s (retention time) of standard was 28.10 min. and all thedesiredpeaksrangedfrom28.06to28.91min.Areaofdesiredpeaks ranged from 25.24 to 220.25 Mau*s (milli- absorbanceunit). Maximum compound percentage was from the Punjab(Semi-aridzone)accession (0.16%)andminimumfromtheAP(Tropicalwetanddryzone)accession(0.03%)ascomparedwithstandard (99%). The percentage of aloe-emodin in differentaccessionshasbeenshownin Figure 4.

DiscussionThe process of investigating plants to identify the chemicalsubstances isofgreat interest toplantscientistsbecausethereisaneedtodiscovernewdrugsfortreatingdiseases.Secondarymetabolitesformedbylivingsystems,notablyfromplantorigin,haveshowngreatpotential in treatinghumandiseasessuchas



cancer,coronaryheartdiseases,diabetesandinfectiousdiseases[29]. These usually consist of the phenolic and polyphenoliccompounds, alkaloids, tannins, saponins, carbohydrates,glycosides, flavonoids and steroids distributed throughout theplantkingdom[30,31].

Aloe vera can grow in almost all types of environmentalconditions but there are several factors that can influence thequality and quantity of a particular constituent. The type ofsolventsandmethodsofpreparationaffectantimicrobialactivityof plants [32]. In present study, we prepared A. vera leaf gelextractsbyusingmethanolasasolvent.Previousstudiesreportthatmethanol as a solvent extracts revealed the antimicrobialproperties of plants due to presence of phenolic compounds,saponin,bryophyllinandothersecondarymetaboliteswhicharereportedtobeantimicrobial[33,34].Methanolicextractinducedthe best extraction yield and more complex composition ofphenolics[35].

Aloe veraplant is rich inalkaloids, tannins,saponins,flavonids,anthraquinones, barbaloins, glycosides and terpenoids [36]. Somethanol is theappropriatesolvent forpreparationofextractsfor antimicrobial study. Our results correlate with previousstudies.Methanol extracts of all 12 accessions exhibited quitegood antimicrobial activity. Previous studies on Aloe vera leafgel extracts showed that extract was more effective againstgrampositivebacteriathangramnegative[37,38].InourstudymethanolicextractshowedgoodactivityagainstgrampositiveS.aureusandgramnegativeE.coliandS.typhi.Ourextractsalsoshowedgoodactivityagainstboththefungalstrainsi.e.A.nigerandC.albicans.

Antimicobial activityAlthough all extracts were quite effective against all thetested strains.But thereare variations indiameterof zonesofinhibition.Solventsystemandextractionprocedurewasuniformthroughout the study.Variation in inhibition zonesmaybe theduetodifferencesinthephytochemicalcompositionofdifferentaccessions. Accessions were from 6 climatic zones of India.

Variations in the sensitivity of the bacterial and fungal speciestested on the extracts might be because of differences in thelocalenvironmentalfactors.Previousstudiesstatesthatmineralandphytochemicalcompositionofplantsisinfluencedbyvariousenvironmental factors including the geography, climate, soilminerals, grazing stress, seasonal changes, phenological stagesandabilityofplantuptakeofmineralsfromsoil[39-41].

AgoodantimicrobialactivitywasalsoshownbyJammuandH.P.accessions.Aloe veraisacoldsensitiveplant.Duringstressmorephytochemicalsareproducedinplantstowithstandtheadverseconditions. Studies conducted on plants in stress conditionsshowed higher production of flavonoids, anthocyanins andmucilaginoussubstancesinstresscondition[42].Flavonoidsandanthocyaninsarebothantimicrobialinnature[43].

Quantification of Aloe- emodinStandardization and characterization of herbal drugs is a topicof continuous scientific interest in the herbal drug industry.With the advent ofmodern chromatographic systems there isan ever increasing intent to produce and develop easy, rapid,convenientandcosteffectivemethodsforstandardization[44].Standardization of methanolic extract of plant leaves requiresHPLCwhichisasensitiveandaccuratetoolwidelyusedforthequalityassessmentofplantextractanditsderivedproductsandformulation[45].TheHighPerformanceLiquidChromatographyrepresents the best technique used now days to separate, tofingerprintqualitativelyandtoidentifyorquantifyeachmoleculefrom complex mixtures as is it the case of plant extracts andherbalsupplements[46].Theuseofmultiplemethodsinvolvingdifferentmobilegradientphaseswouldincreasethevalidityandreliabilityoftheobtainedresults.

Therearenottoomuchpreviouscomparativestudiesondiversitybases which can correlates the marker compound analysis ofAloe vera. The results indicated that,Aloe vera plant collectedfrom humidsubtropical and semi- arid zone showed betteraloe- emodin production as compared to tropical area zone.Cassia, another plant genus which is a important source of

Accessions CA AN SF EF SA PM ST SM KP EC PAJammu 1.2 2.5 2.5 2.5 1.2 2.5 1.2 1.2 2.5 1.2 2.5HP 1.2 2.5 2.5 2.5 1.2 2.5 1.2 1.2 2.5 1.2 2.5Panjab 2.5 5.0 2.5 2.5 1.2 5.0 2.5 2.5 2.5 2.5 5.0Haryana 2.5 5.0 2.5 2.5 1.2 5.0 2.5 1.2 2.5 1.2 5.0Rajasthan 2.5 5.0 2.5 2.5 1.2 5.0 1.2 1.2 2.5 1.2 5.0Gujarat 5.0 5.0 5.0 2.5 5.0 5.0 2.5 5.0 2.5 2.5 5.0UP 1.2 5.0 5.0 2.5 1.2 5.0 2.5 1.2 2.5 1.2 5.0MP 2.5 5.0 2.5 2.5 2.5 5.0 2.5 5.0 2.5 1.2 5.0WB 5.0 5.0 2.5 2.5 1.2 5.0 1.2 2.5 2.5 1.2 5.0AP 2.5 5.0 5.0 2.5 5.0 5.0. 2.5 5.0 2.5 2.5 5.0Goa 5.0 5.0 5.0 2.5 1.2 5.0 1.2 5.0 2.5 1.2 5.0Kerala 5.0 5.0 5.0 2.5 1.2 5.0 1.2 2.5 2.5 1.2 2.5Fungalstrains=CA-Candida albicans andAN-Aspergillus nigerBacterialstrains=EC- Escherichia coli,PM- Proteus mirabilis,KP-Klebsiella pneumoniae,PA-Pseudomonas aeruginosa,SF-Shigella flexneri,SM-Serratia marcescens,ST-Salmonella typhi,EF-Enterococcus faecalisandSA- Staphylococus aureus

Table 4 MinimumInhibitoryConcentration(MICinmg/ml)ofvariousaccessionsofAloe veraExtractsagainstdifferentmicro-organisms



Agro-climatic zones Sample Name RT (min.) Area (Mau*s) Yield (%)Highland

Jammu 28.21 112.23 0.0826HimachalPradesh(HP) 28.06 107.82 0.0793

Semi-Arid

Punjab 28.21 220.25 0.1621Haryana 28.91 129.11 0.095

Arid

Rajasthan 28.13 27.84 0.0205Gujarat 28.17 25.24 0.0186

Humid-Subtropical

UttarPradesh(UP) 28.13 154.15 0.1134MadhyaPradesh(MP) 28.17 80.35 0.0591

Tropicalwetanddry

WestBengal(WB) 28.18 91.15 0.0671AndhraPradesh(AP) 28.2 45.83 0.0337

Tropicalwet

Goa 28.22 204.53 0.1505Kerala 28.25 99.88 0.0735

Standard Aloe-emodin 28.1 135779 99

Table 5 HPLCanalysisofdifferentAloe veraaccessions

HPLCchromatogramofStandardFigure 3a

naturallyoccurringbioactivecompoundsanthraquinonesalsofoundintropicalandsubtropicalareasthroughouttheworld[47]. Itsupports thestatementthatsubtropicalenvironmentconditions favor the anthraquinones production from theplants. It is known that genetic and environmental factorsand their interactions affect the pharmaceutically importantsecondarymetabolites inmedicinal plants [48]. A variety ofenvironmental factors; such as altitude, radiation, and soilnutrition; have been proven to significantly influence thesecondarymetaboliteprofile[49,50].Furthermore,soiltype,sun exposure, temperature and rain fall also have majoreffectsonthephenolcontentofplants.Thevariationsintheanthraquinoneglycosidecontentcouldbeaffectedduetothedifferentaltitudewheretheplantwasgrown[51].

Modern world is moving back towards herbal drugs.But standardization of herbal drugs with respect toconcentration of its active principles is a must for theirapprovalinmodernmedicinesystem.Wehavestandardizedan antimicrobial flavanol aloe- emodin from methanolicextractsofAloe vera.Presentstudyemphasizesthatamountof aloe- emodin in extracts which may be related to theantimicrobialactivityshown.Punjab,Haryana,UPandWestBengalshowedsignificantcorrelationbetweenpercentageofpresenceofmarkercompoundandantimicrobialactivity.Moremarkerantimicrobialcompoundsshouldbescreenedfor formulationofanherbaldrugwithwideracceptabilityfrom Aloe vera.Itwillhelpincommercializationofplantinparticularareaswithdesiredpharmacologicaleffects.



HPLCchromatogramofGujarataccessionFigure 3b

HPLCchromatogramofUttarPradeshaccessionFigure 3c

ConclusionAloe vera isaplantofgreat interest for the researchpurposesbut most of works have been done on pharmaceutical level.Phytochemical composition of plants is influenced by variousenvironmental and climatic factors. Thepresent studyhas also

showed the role of climatic conditions at different geographicallocations on the presence of bioactive component aloe- emodinin crude extracts of Aloe vera due to which it may have goodantibacterialactivity. It ishopedthatthisstudywould leadtotheestablishmentofsomecompoundsthatcouldbeusedtoformulatenewandmorepotentantimicrobialdrugsofnaturalorigin.



Conflict of InterestThereisnoconflictofinterestbetweenauthors.

AcknowledgementFinancialAssistancefromUGC,NewDelhiintheformofUGC-SAPgrant(No.F.3-20/2012,SAPII)andUGC-BSRfellowshipisgratefullyacknowledged.

HPLCchromatogramofUttarPradeshaccessionFigure 3d

Figure 4 Showingtheyieldpercentageofaloe-emodinfromdifferentaccessions



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